The role of high cholesterol-containing microdomains in the signal transduction cascade leading to the activation of volume-regulated anion channels (VRACs) was studied.Methods
Osmotic cell swelling-induced efflux of 125I− was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann–Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre-incubation with 2-hydroxypropyl-β-cyclodextrin in the presence of acceptor lipid vesicles.Results
Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume-sensitive anion efflux was markedly inhibited when cholesterol-loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin-1 deficient Caco-2 colonocytes as well as in sphingomyelinase-treated Intestine 407 cells, indicating that cholesterol-rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1-dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity-provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume-sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts.Conclusions
The results support a model of regulatory volume decrease involving recruitment of volume-sensitive anion channels from intracellular compartments to the plasma membrane.